tailless

EVOLUTIONARY HOMOLOGS

A comparative tree of DNA-binding domain amino acid sequences reveals the evolutionary affinities of Drosophila nuclear receptor proteins. Knirps shows no close affinities to other nuclear receptor proteins. Drosophila Ecdysone receptor sequence is most similar to murine RIP14. Tailless has a close affinity to murine Tlx. Drosophila E78 and E75 fall in the same subclass as Rat Reverb alpha and beta, and C. elegans "CNR-14." Drosophila HR3 is in the same subclass as C. elegans "CNR-3." Drosophila HNF-4 is most closely related in sequence to Rat HNF-4. Drosophila Ftz-F1 and Mus ELP show sequence similarity to each other. Drosophila Seven up is closely related to Human COUP-TF. Drosophila Ultraspiracle is in the same subfamily as Human RXRalpha, Human RXRbeta, and Murine RXRgamma. The latter two groups, containing Ultraspiracle and Seven up, show a distant affinity to each other. Four other subfamilies show no close Drosophila affinities. These are: 1) C. elegans rhr-2, 2) Human RARalpha, beta and gamma, 3) Human thyroid hormone receptor alpha and beta, and 4) Human growth hormone receptor, glucocorticoid receptor, and progesterone receptor (Sluder, 1997).

There is high sequence similarity between Drosophila Dissatisfaction and the vertebrate Tailless proteins in both the DNA binding domain (DBD) and ligand binding domain (LBD). The greatest similarity between Dsf and vertebrate Tailless is in the DBD and adjacent T box region, with an overall identity of 81%, including 100% identity in the P box and T box sequences. The human, mouse, and chicken Tailless LBD sequences are rated most similar to Dsf, with 44% amino acid identity. The degree of similarity between Dsf and the vertebrate Tailless proteins in the LBD is greater than the similarity between human Tailless and any protein that is not a vertebrate Tailless protein. The LBD of Drosophila Tailless is related at a substantially lower degree (35% identity). In addition to the high degree of similarity in sequence in the DBDs and LBDs, DSF and Tailless proteins are similar in having relatively short amino-terminal and carboxy-terminal sequences preceding the DBDs or following the LBDs. Dsf diverges from all Tailless proteins within the D box region of the DBD and in the A box region that follows the DBD and T box sequences. Dsf also contains a notably longer sequence (380 amino acids) linking the DBD with the LBD that has no similarity to Tailless proteins or to any other proteins in GenBank (Finley, 1998 and references).

To infer similarities and differences in terminal pattern formation in insects, several of the key genes of this process were analyzed in the beetle Tribolium castaneum. Two genes of the terminal pattern cascade, namely tailless (tll) and forkhead (fkh), from Tribolium were cloned and their expression patterns were studied. In addition, the pattern of MAP kinase activation was analyzed at blastoderm stage as a possible signature for torso-dependent signaling. Further, the late expression of the previously cloned Tribolium caudal (Tc-cad) gene was examined. Finally, the upstream region of Tc-tll was used to drive a reporter gene construct in Drosophila. This construct is activated at the terminal regions in Drosophila, suggesting that the torso-dependent pathway is conserved between the species. Most of the expression patterns of the genes studied here are similar in Drosophila and Tribolium, suggesting conserved functions. There is, however, one exception, namely the early function of Tc-tll at the posterior pole. In Drosophila, the posterior tll expression is involved in the direct regulation of the target genes of the terminal pathway. In Tribolium, posterior Tc-tll expression occurs only for a short time and ceases before the target genes known from Drosophila are activated. Thus, it is inferred that Tc-tll does not function as a direct regulator of segmentation genes at the posterior end. It is more likely to be involved in the early specification of a group of 'terminal' cells, which begin to differentiate only at a later stage of embryogenesis, when much of the abdominal segmentation process is complete. Thus, there appears to have been a major shift in tll function during the evolutionary transition from short germ to long germ embryogenesis (Schroder, 2000).

There are at least three known direct target genes of tll that are required for the formation of the posterior terminal structures of the Drosophila embryo, namely fkh, byn, and the terminal hb stripe. All three are thought to be regulated directly by tll, although this has been shown formally only for the terminal hb stripe. In Tribolium, all three of these genes (or expression domains) start to be expressed only after tll expression has ceased at the posterior end. This late onset of expression of the target genes suggests that tll could not be their direct activator. A similar inference applies to another target gene of tll, namely Krüppel (Kr). tll acts as a repressor of Kr in Drosophila. In Tribolium, Tc-Kr expression occurs at the posterior pole of the blastoderm embryo, overlapping with the early Tc-tll expression. This coexpression suggests that Tc-tll does not act as a repressor of Tc-Kr. Thus, Tc-tll does not appear to play the same role as tll in Drosophila as a direct regulator of other segmentation genes (Schroder, 2000).

Nonetheless, Tc-tll is likely to be involved in determining the posterior terminal fate in Tribolium. This can be inferred from the observations presented here, as well as from classic fate mapping experiments. A receptor tyrosine kinase pathway is active at the same time and location where Tc-tll is activated at the posterior pole, suggesting that the torso-mediated induction of tll is conserved between the two species. Accordingly, a reporter gene construct carrying the Tc-tll upstream region in Drosophila is activated in a very similar pattern as that known for the endogenous tll pattern in Drosophila. The other clue comes from the general fate mapping experiments in short germ insects. These fatemaps suggest that the future terminus, represented by the hindgut anlage, is specified already at blastoderm stage, although the structure itself develops only much later. This is in contrast to the abdominal segments, for which there is no representation in the blastoderm fate map. They are sequentially generated from the posterior growth zone only after blastoderm stage. To reconcile these observations, it is proposed that there is a group of cells within the growth zone that is determined at blastoderm stage to produce the terminal structures, but which remains quiescent until much of the abdominal segmentation has been completed. In this scenario, the most likely role for Tc-tll would be an involvement in the differentiation of cells that could be called 'terminal cells' (Schroder, 2000).

Specification of neuron identity requires the activation of a number of discrete developmental programs. Among these is pathway selection by growth cones: in order for a neuron's growth cone to respond appropriately to guidance cues presented by other cells or the extracellular matrix, the neuron must express genes to mediate the response. The fax-1 gene of C. elegans is required for pathfinding of axons that extend along the ventral nerve cord. fax-1 is also required for pathfinding of axons in the nerve ring, the largest nerve bundle in the nematode, and for normal expression of FMRFamide-like neurotransmitters in the AVK interneurons. The fax-1 gene encodes a member of the superfamily of nuclear hormone receptors and has a DNA-binding domain related to the human PNR and Drosophila Tailless proteins. fax-1 expression is observed in embryonic neurons, including the AVK interneurons, just prior to axon extension, but after neurogenesis. These data suggest that fax-1 coordinately regulates the transcription of genes that function in the selection of axon pathways, neurotransmitter expression and, perhaps, other aspects of the specification of neuron identity (Much, 2000).

The chordate central nervous system has been hypothesized to originate from either a dorsal centralized, or a ventral centralized, or a noncentralized nervous system of a deuterostome ancestor. In an effort to resolve these issues, the hemichordate Saccoglossus kowalevskii was examined and the expression of orthologs of genes that are involved in patterning the chordate central nervous system was examined. All 22 orthologs studied are expressed in the ectoderm in an anteroposterior arrangement nearly identical to that found in chordates. Domain topography is conserved between hemichordates and chordates despite the fact that hemichordates have a diffuse nerve net, whereas chordates have a centralized system. It is proposed that the deuterostome ancestor may have had a diffuse nervous system, which was later centralized during the evolution of the chordate lineage (Lowe, 2003).

The adult S. kowalevskii has tripartite, tricoelomic organization. At the anterior is the muscular proboscis or prosome, used for burrowing and collecting food particles. It contains the heart, kidney, a section of the dorsal nerve cord, and the protocoel. The middle region, which is the collar or mesosome, contains the mouth, a section of dorsal nerve cord formed by neurulation, the paired mesocoels, and the base of the stomochord, which projects forward into the prosome. The posterior region or metasome contains the gill slits, the remainder of the dorsal nerve cord, the entire ventral nerve cord, paired metacoels, gonads, a long through-gut, and terminal anus. At juvenile stages, a ventral post-anal extension (called a tail or sucker) is present (Lowe, 2003).

Gastrulation entails uniform and simultaneous inpocketing of the vegetal half of the hollow blastula. As the blastopore closes, a gumdrop-shaped gastrula is formed. As the embryo lengthens, two circumferential grooves indent and divide the length into prosome, mesosome, and metasome regions. Mesodermal coeloms outpouch from the gut anteriorly and laterally. The first gill slit pair appears externally by day 5, and the animal bends from the dorsal side. The hatched juvenile elongates and adds further pairs of gill slits successively. The animal is nearly bilaterally symmetric, except that the prosome excretory pore (the proboscis pore) from the kidney is reliably on the left, defining a left-right asymmetry (Lowe, 2003).

The hemichordate adult nervous system is not centralized but is a diffuse intraepidermal, basiepithelial nerve net. Nerve cells are interspersed with epidermal cells and account for 50% or more of the cells in the proboscis and collar ectoderm and a lower percentage in the metasome. Axons form a meshwork at the basal side of the epidermis. The two nerve cords are through-conduction tracts of bundled axons and are not enriched for neurogenesis. This general organizational feature of the nervous system has been largely underemphasized in recent literature that focuses on possible homologies between chordate and hemichordate nerve cords (Lowe, 2003).

Twenty-two full-length coding sequences of orthologs associated with neural patterning in chordates were isolated. These genes are probably present as single copies in S. kowalevskii because orthologs of most of them are present as single copies in lower chordates and echinoderms, and many of the genes were recovered multiple times in the EST analysis without finding any closely related sequences (Lowe, 2003).

Using full-length probes for in situ hybridization, all 22 genes were found to be expressed strongly in the ectoderm as single or multiple bands around the animal, in most cases without dorsal or ventral differences (rx, hox4, nkx2-1, en, barH, lim1/5, and otx are exceptions). Circumferential expression is consistent with diffuse neurogenesis in the ectoderm. The domains resemble the circumferential expression of orthologs in Drosophila embryos. In chordates, by contrast, most of these neural patterning genes are expressed in stripes or patches only within the dorsal neurectoderm and not in the epidermal ectoderm. Also, in chordates, the domains are often broader medially or laterally within the neurectoderm, and there are usually additional expression domains in the mesoderm and endoderm. In most of the 22 cases in S. kowalevskii, the ectodermal domain is the only expression domain (six3, otx, gbx, otp, nkx2-1, dbx, hox11/13, and irx are exceptions) (Lowe, 2003).

Although each of the 22 genes has a distinct expression domain along the anteroposterior dimension of the chordate body, attempts were made to divide them into three broad groups to facilitate the comparison with hemichordates: anterior, midlevel, and posterior genes. Anterior genes are those which in chordates are expressed either throughout or within a subdomain of the forebrain. Midlevel genes are those expressed at least in the chordate midbrain, having anterior boundaries of expression in the forebrain or midbrain, and posterior boundaries in the midbrain or anterior hindbrain. Posterior genes are those expressed entirely within the hindbrain and spinal cord of chordates. Many of the chordate genes have additional domains of expression elsewhere in the nervous system and in other germ layers, but comparisons were restricted to domains involved in specifying the neuraxis in the anteroposterior dimension. Taking these groups of genes one at a time, it was asked where the orthologous genes are expressed in S. kowalevskii. In all comparisons, no morphological homology is implied between the subregions of the chordate and hemichordate nervous systems (Lowe, 2003).

Ten genes expressed in midlevel neural domains were examined, namely tailless (tll), paired box homeobox 6 (pax6), emptyspiracles-like (emx), barH, orthopedia (otp), developing brain homeobox (dbx), lim domain homeobox 1/5 (lim1/5), iroquois (irx), orthodenticle-like (otx), and engrailed (en). These genes are all expressed in chordates at least in the midbrain of the central nervous system, and thus, as a group, their domains are more posteriorly located than the anterior set. Some have the anterior border of the domain in the forebrain (tll, pax6, emx, lim1/5, and otx), and some have their anterior border in the midbrain (otp, barH, dbx, irx, and en). Most have posterior borders in the midbrain, but two (en and irx) have posterior borders in the anterior hindbrain. Thus, while all are expressed in the midbrain, each differs in its anterior and posterior extent. Several of the chordate genes (pax6, dbx, en, and irx) have separate posterior expression domains running the length of the chordate hindbrain and spinal cord at different dorsoventral levels of the neural tube (Lowe, 2003).

In S. kowalevskii, these ten orthologs are expressed in circumferential bands in the ectoderm at least of the mesosome (collar) or anterior metasome, that is, more posteriorly than the anterior group. Each gene differs in the exact anteroposterior extent of its domain -- some are expressed in part or all of the prosome. The most broadly expressed orthologs of this group are pax6, otp, lim1/5, irx, and otx. All are expressed in the prosome (relatively weakly for otx), mesosome (weakly in the case of otp and lim1/5), and anterior metasome, all ceasing by the level of the first gill slit. pax6 is strongest at the base of the proboscis, and lim1/5 is expressed most strongly in a dorsal patch at the base of the proboscis. The most narrowly expressed orthologs are barH, tll, emx, and en. tll is detected in early stages in the anterior prosome, posterior prosome, and anterior mesosome and in later stages restricted to the anterior mesosome. The emx domain is a single ring in the anterior mesosome plus an additional domain in the ciliated band in the posterior metasome, the only gene of the 25 to be expressed in the band cells. barH and en are both expressed in narrow ectodermal bands; barH in the anterior mesosome and en in the anterior metasome. A dorsal view of both en and barH reveals a dorsal narrow gap in expression in the midline. Ventrally, no such gap is observed. Two additional spots of en expression are detected in the ectoderm on either side of the dorsal midline in the proboscis. In the most posterior ring of otx expression in the metasome, a similar gap in expression is observed. otp is expressed predominantly in a punctate pattern in the apical layer of prosome ectoderm and in a diffuse pattern in the basal layer of prosome ectoderm, similar to dlx. It is also expressed in a circumferential ring of intermittant ectodermal cells in the posterior mesosome and then in two parallel lines of cells bilateral to the dorsal axon tract of the anterior metasome. Early dbx expression is most strongly detected in an ectodermal ring in the developing mesosome overlapping the posterior domain of tll. dbx is also expressed in the prosome at low levels throughout the ectoderm and at high levels in scattered individual cells or groups of cells. Later expression is restricted to two ectodermal bands marking the anterior and posterior limits of the mesosome. An additional endodermal domain of expression is observed predominantly in the ventral anterior pharyngeal endoderm (Lowe, 2003).

otx, en, and irx deserve description in more detail because in chordates, especially vertebrates, the products of these regionally expressed genes are thought to interact in setting up the midbrain-hindbrain boundary and the isthmic organizer. Furthermore, the otx domain at the midbrain level is the site from which neural crest cells migrate ventrally to the first branchial arch. In S. kowalevskii, otx is expressed at low but readily detectable levels in the prosome ectoderm and at high levels in four closely spaced ectodermal rings: one at the base of the prosome, two in the mesosome, and one in the anterior metasome. This fourth stripe of otx expression crosses the site where the first gill slit perforates the ectoderm. As evidence, beyond morphology, that the hemichordate gill slit is homologous to the chordate gill slit/branchial arch, the pax1/9 ortholog, known to be expressed in chordate gill slits, is expressed in the endoderm of the developing S. kowalevskii gill slit. Gill slit expression of pax1/9 is observed in the adult of P. flava. Thus, chordates and hemichordates have in common the association of the posterior limit of the otx domain with the position of the first gill slit or branchial arch (Lowe, 2003).

In hemichordates, the en domain overlaps the posterior part of the otx domain, and the irx domain runs through both of these, as is also the case in chordates. However, otx expression in S. kowalevskii extends slightly more posteriorly than does en, whereas in chordates the en domain extends slightly more posteriorly (Lowe, 2003).

In summary, for this midlevel group of genes, the S. kowalevskii orthologs are expressed in the mesosome and anterior metasome (with some domains extending anteriorly into the prosome), that is, more posteriorly than those genes of the anterior group. In general, expression domains that end posteriorly near the midbrain-hindbrain boundary in chordates, end in the anterior metasome in hemichordates. Although the anterior metasome is not the site of an obvious morphological boundary, it is the site of the first gill slit. The first gill slit/branchial arch in chordates is at the same body level as the midbrain-hindbrain boundary (Lowe, 2003).

The 22 expression domains of orthologs of chordate neural patterning genes of S. kowalevskii correspond strikingly to those in chordates. There are differences such as the extent of overlap of edges of domains of otx, en, and gbx and other midlevel genes that are critical for forming boundaries within the chordate brain, but the relative domain locations are nonetheless very similar. This similar topography of domains is most parsimoniously explained by conservation in both lineages of a domain arrangement (a map) already present in the common ancestor, the ancestor of deuterostomes (Lowe, 2003).

At least 14 of the 22 conserved domains have similar locations in one or more protostome groups. Such similarities are most parsimoniously explained as a conservation of domains from the ancestral bilaterian. In the case of the hox genes, otx, emx, pax6, six3, gbx, and tll, there is strong evidence for such conservation, but less so for the others (barH and rx). At least four of the chordate-hemichordate conserved domains may not be shared by protostomes. Namely, three of these genes (dbx, vax, and hox11/13) are absent from the Drosophila genome and have not been cloned from other protostome groups. Also, one gene, engrailed, has no clear corresponding domain of expression known in protostomes. In Drosophila, en is expressed in the posterior compartments of 14 body segments and at three or more sites in the head that probably derive from ancient preoral segments. This pattern for en appears very different from the single ectodermal band in deuterostomes (Lowe, 2003).

The nerve net of hemichordates could represent the basal condition of the deuterostome ancestor, or it could represent the secondary loss of a central nervous system from an ancestor. Was the complex map of the ancestor associated with a complex diffuse nerve net or a central nervous system in the ancestor? It is suggested that the deuterostome ancestor may have had a diffuse basiepithelial nervous system with a complex map of expression domains, though not necessarily a diffuse net exactly like that of extant hemichordates. Hemichordates would then have retained a diffuse system in their lineage and early in the chordate lineage, centralization would have taken place. In this proposal, the domain map predates centralization and is carried into the nervous system. In this respect, the core questions of nervous system evolution would concern the modes of centralization utilized by the ancestor's various descendents rather than a dorsoventral inversion, per se. Thus, it is proposed that in chordates, especially vertebrates, the major innovation may have been the formation of a large contiguous nonneural (epidermogenic) region (Lowe, 2003).

A unique vertebrate nuclear receptor, Tlx, is expressed exclusively in the neuroepithelium of the embryonic brain. Sequence comparison reveals similarity to tailless (tll). Tlx and Tll proteins share a target gene specificity that is unique among the nuclear receptor superfamily. Ectopic expression of Tlx in fly embryos causes a repression of segmentation comparable to that elicited by Tll. The similarities in structure, expression pattern, target gene specificity and phenotypes in transgenic flies suggest conservation of genetic programs upstream and downstream of this Tlx/Tll class of nuclear receptors during embryogenesis (Yu, 1994).

A mouse homolog of the Drosophila tailless gene shows considerable homology in the DNA-binding domain, suggesting that the respective proteins bind similar recognition sequences. Although the ligand-binding domain shares features in common with the Tailless ligand domain, it also shares conserved amino acid stretches with other orphan nuclear receptors: human ovalbumin upstream binding protein transcription factors (hCOUP-TF I and II). The expression of tailless in mice is specifically localised to the developing forebrain from day 8 p.c. and in dorsal midbrain from day 8.75 p.c. The expression pattern of tailless has been compared to that of other forebrain markers, including distal-less (Dlx1), brain factor 1 (BF1), and the orthodenticle genes (Otx1 and Otx2). In addition to the developing forebrain, these genes show dynamic patterns of expression in two structures whose development requires inductive signals from the forebrain: the eye and the nose. Any posterior activity for the mouse tailless homolog is not evident (Monaghan, 1995).

The gene tailless is a member of the superfamily of genes that encode transcription factors of the ligand-activated nuclear receptor type, and is expressed in the invertebrate and vertebrate brain. In mice, its transcripts are restricted to the periventricular zone of the forebrain, the site of origin of neurons and glia. Homologous recombination was used to generate mice that lack a functional tailless protein. Homozygous mutant mice are viable at birth, indicating that tailless is not required for prenatal survival; however, adult mutant mice show a reduction in the size of rhinencephalic and limbic structures, including the olfactory, infrarhinal and entorhinal cortex, amygdala and dentate gyrus. At E15.5, mutant mice show a reduction in the number of cells in the intermediate zone of the cortex, consistent with a role for the tlx gene in ventricular zone proliferation and differentiation. A reduction in the size of the olfactory bulb and rhinencephalic ventricular zone is also evident. Both male and female mice are more aggressive than usual and females lack normal maternal instincts. These animals therefore enable a molecular approach to be taken towards understanding the genetic architecture and morphogenesis of the forebrain (Monaghan, 1997).

The Xenopus homolog Xtll of the Drosophila gene tailless (tll), which is expressed during early eye development, is characterized with respect to its relationship to vertebrate regulators of eye morphogenesis. Overall amino acid identities are 91% between Xtll and mouse tll and 39% in a comparison with Drosophila tll. Xtll expression is first detected in open neural fold stage (stage 16) embryos within an area located in the anterior, prechordal portion of the neural plate. Two groups of cells lateral to the most anterior portion of the neural groove exhibit Xtll-specific transcripts. These two patches of cells are connected by a thin line of Xtll-positive cells over the median midline. Lineage analysis of the same cells demonstrates that they will give rise to the eyes and chiasmatic ridge of early tadpoles. Expression of Xtll, Pax6 and Rx is first detected in the area corresponding to the eye anlagen within the open neural plate in partially overlapping, but not identical, patterns. During the evagination of the optic vesicle, Xtll expression is most prominent in the optic stalk, as well as in the distal tip of the forming vesicle. In tadpole-stage embryos, Xtll gene transcription is most prominent in the ciliary margin of the optic cup. Inhibition of Xtll function in Xenopus embryos interferes specifically with the evagination of the eye vesicle and, in consequence, Xpax6 gene expression is severely reduced in such manipulated embryos. These findings suggest that Xtll serves an important regulatory function in the earliest phases of vertebrate eye development (Hollemann, 1998).

Although the development of the vertebrate eye is well described, the number of transcription factors, known to be key to this process, is still limited. The localized expression of the orphan nuclear receptor Tlx (a homolog of Drosophila Tailless) in the optic cup and discrete parts of the central nervous system suggests the possible role of Tlx in the formation or function of these structures. Analyses of Tlx targeted mice reveal that, in addition to the central nervous system cortical defects, lack of Tlx function results in progressive retinal and optic nerve degeneration with associated blindness. An extensive screen of Tlx-positive and Tlx-negative P19 neural precursors has identified Pax2 as a candidate target gene. This identification is significant, because Pax2 is known to be involved in retinal development in both the human and the mouse eye. Pax2 is a direct target and the Tlx binding site in its promoter is conserved between mouse and human. These studies show that Tlx is a key component of retinal development and vision and an upstream regulator of the Pax2 signaling cascade (Yu, 2000).

The C. elegans tailless/Tlx homolog nhr-67 regulates a stage-specific program of linker cell migration in male gonadogenesis

Cell migration is a common event during organogenesis, yet little is known about how migration is temporally coordinated with organ development. Stage-specific programs of cell migration are being investigated using the linker cell (LC), a migratory cell crucial for male gonadogenesis of C. elegans. During the L3 and L4 larval stages of wild-type males, the LC undergoes changes in its position along the migratory route, in transcriptional regulation of the unc-5 netrin receptor and zmp-1 zinc matrix metalloprotease, and in cell morphology. The tailless homolog nhr-67 was identified as a cell-autonomous, stage-specific regulator of timing in LC migration programs. In nhr-67-deficient animals, each of the L3 and L4 stage changes is either severely delayed or never occurs, yet LC development before the early L3 stage or after the mid-L4 stage occurs with normal timing. It is proposed that there is a basal migration program utilized throughout LC migration that is modified by stage-specific regulators such as nhr-67 (Kato, 2009).

This study describes how the C. elegans male LC relies on timing cues to execute the various stages of its migration. Closer examination of LC migration revealed that the LC displays many complex behaviors, including migration over different body surfaces, the execution of two turns, and changes in cell shape and migration speed. Moreover, these behaviors occur at specific times during the migration, suggesting that dynamic, stage-specific gene regulation is involved. Three genes that were found to exhibit stage-specific expression in the LC are the tailless homolog nhr-67, the netrin receptor unc-5 and the zinc metalloprotease zmp-1. nhr-67 is expressed by the LC during the L3 and L4 stages and is discussed below. unc-5 is expressed from the late L2 stage to the mid-L3 stage, and its downregulation is necessary for the LC to turn ventrally in the mid-L3 stage. unc-5 is likely to be just one of several ECM receptors dynamically regulated in the LC, as judged by the fact that other migratory cells, such as the hermaphrodite DTCs in C. elegans and the neural crest and primordial germ cells in vertebrates, express several different ECM receptors required to navigate their complex course. zmp-1 is expressed only in the L4 stage. No defect was foubd in LC migration in zmp-1 deletion mutants, but this might be due to redundancy of matrix metalloproteases in the LC. In addition to these three genes, stage-specific cell behaviors, such as the L4 stage LC shape change, were uncovered that are not mediated by UNC-5 or ZMP-1, suggesting that the LC expresses many other genes stage-specifically (Kato, 2009).

One way to modulate complex cell behaviors is through instructional cues that are spatially restricted, such that a cell modifies its behavior upon reaching specific sites along its migratory route. Instead, the LC appears to implement these behaviors as part of its developmental program based on a temporal cue. It was shown, for instance, that changes in cell shape and zmp-1 expression occur in L4 larvae even when the LC has not reached its normal position in the posterior body (Kato, 2009).

This study has revealed a new function for nhr-67 in controlling a time-dependent subset of events during LC migration. nhr-67 acts cell-autonomously to regulate all the LC changes that have been identified within a specific time-window of the L3 and L4 stages, but none of the migratory changes either before or afterwards. This observation suggests that LC migration is assembled from temporal subprograms that might each be independently regulated by factors such as nhr-67 (Kato, 2009).

In addition, nhr-67 controls the timing of events during the subprogram it regulates. For each of the developmental events in the LC during the L3 and L4 stages, nhr-67(RNAi) animals display a phenotype indicative of the continuation of the early L3 stage and the delay of succeeding stages. There is evidence that Tlx, the mammalian homolog of nhr-67, regulates timing in mouse neurogenesis. In nhr-67(RNAi) animals, UNC-5, which in wild-type animals is downregulated in the LC by the mid-L3 stage, continues to be highly expressed in the late L3 stage and during the L4 stage, whereas zmp-1, a gene normally expressed in the LC at L4 stage, is never expressed. These roles of NHR-67 are consistent with previous findings that tailless acts as both a positive and negative regulator of gene expression. Also, in nhr-67(RNAi) animals, the LC does not turn from the dorsal to the ventral bodywall during the mid-L3 stage as in wild-type worms. This abnormal guidance is due, at least in part, to the continued expression of UNC-5 in nhr-67(RNAi) animals past the mid-L3 stage. Finally, in contrast to the increasingly polarized morphology of the LC in wild-type worms, the LC of nhr-67(RNAi) males remains in the round, early L3 stage shape throughout most of its migration. Although an nhr-67 null mutant might have a more severe phenotype than nhr-67(RNAi), the nhr-67(RNAi) phenotype is both penetrant and consistent in each animal and across trials (Kato, 2009).

An interesting question is how NHR-67 regulates events in the LC at diverse times during the L3 and L4 stages. One possibility is that NHR-67 confers specificity by binding to a heterodimeric partner or co-regulator. Another possibility is that a temporal gradient is set by the level of expressed NHR-67, which would gradually accumulate over time. An example of this is seen with PHA-4, the master regulator of pharynx formation, which first activates genes whose regulatory regions have the highest affinity for PHA-4, and later activates genes whose regulatory regions have lower affinity (Kato, 2009).

It is proposed that LCs have both a basal migration program, which begins at LC specification and is used through the life of the LC, and at least three stage-specific programs that modify the basal program. NHR-67 regulates one such program from the early L3 to mid-L4 stage. Since in nhr-67(RNAi) animals, LC migration before the early L3 stage and after the mid-L4 stage occurs with normal timing, there must be other transcriptional regulators acting in the LC. It is hypothesized that each of these stage-specific regulators acts on a basal migration program consisting of genes that are expressed throughout LC migration. Some of the genes that were examined are part of the basal migration program, including lag-2, gon-1, him-4 and mig-2; the latter three are required for normal gonadal migration. Since the expression of these genes was unaffected in nhr-67(RNAi) animals, it is proposed that the basal migration program continues even without the execution of stage-specific programs (Kato, 2009).

Cell migrations are often required during organogenesis and contribute to the shape and function of the final organ. In many of these cell migrations, spatially graded cues not only provide guidance but also regulate motility, cell morphology and gene expression. For example, in border cell migration in Drosophila, the migratory behavior is mediated through EGF and PDGF/VEGF receptors that bind signaling molecules from a spatially restricted origin. Similarly, tracheal outgrowth in mouse or Drosophila requires FGF signaling from surrounding tissue, with the cells closest to the tissues being activated. Temporal regulation of migration is less well characterized, perhaps because spatial cues have such a visible role in most cell migrations. Unlike other well-studied instances of cell migration, this study has shown that the migration of the LC depends heavily on timing cues to execute different stages of its migration. It was found that even when the LC is mispositioned in the body of the animal, and hence removed from its normal spatial environment, it is still capable of migrating and undergoing stage-specific programs. The delayed development of the LC in nhr-67(RNAi) animals also suggests that the LC uses NHR-67 to interpret timing information, the source of which might be a cell-intrinsic clock or a global timing cue. One hint that a global signal may be involved comes from the previous finding that the DAF-12 nuclear hormone receptor, which binds the global hormone dafachronic acid, is required by both the male LC and hermaphrodite DTCs for executing gonadal turns in L3 larvae and for arresting development in dauer larvae (Kato, 2009).

Understanding temporal regulation during organogenesis should shed light on morphological differences between species, and between normal and diseased states. Since the gonads of different nematode species have different shapes, it is interesting to speculate that dramatic changes in gonadal shape could arise from subtle changes in temporal regulation of the LC. The existence of stage-specific regulators suggests the possibility that dramatic changes in organ shape can be achieved through the regulation of relatively few temporally regulated genes (Kato, 2009).

The C. elegans Tailless/TLX transcription factor nhr-67 controls neuronal identity and left/right asymmetric fate diversification

An understanding of the molecular mechanisms of cell fate determination in the nervous system requires the elucidation of transcriptional regulatory programs that ultimately control neuron-type-specific gene expression profiles. This study shows that the C. elegans Tailless/TLX-type, orphan nuclear receptor NHR-67 acts at several distinct steps to determine the identity and subsequent left/right (L/R) asymmetric subtype diversification of a class of gustatory neurons, the ASE neurons. nhr-67 controls several broad aspects of sensory neuron development and, in addition, triggers the expression of a sensory neuron-type-specific selector gene, che-1, which encodes a zinc-finger transcription factor. Subsequent to its induction of overall ASE fate, nhr-67 diversifies the fate of the two ASE neurons ASEL and ASER across the L/R axis by promoting ASER and inhibiting ASEL fate. This function is achieved through direct expression activation by nhr-67 of the Nkx6-type homeobox gene cog-1, an inducer of ASER fate, that is inhibited in ASEL through the miRNA lsy-6. Besides controlling bilateral and asymmetric aspects of ASE development, nhr-67 is also required for many other neurons of diverse lineage history and function to appropriately differentiate, illustrating the broad and diverse use of this type of transcription factor in neuronal development (Sarin, 2009).

The function of nhr-67 is not restricted to the nervous system. In the developing vulva, nhr-67 controls the identity of several vulval cell types, acting in conjunction with several distinct regulatory factors. As is the case in the ASE neurons, nhr-67 acts within the context of a bistable system in which two factors, cog-1 and nhr-67, cross-inhibit the activity of one another to generate distinct vulval cell types. The interaction of nhr-67 and cog-1 in ASE neurons is, however, strikingly distinct from the interaction in the vulva, because in ASE nhr-67 activates cog-1, whereas in the vulva it inhibits its expression, presumably because it co-operates with factors other than CHE-1 to control cog-1 expression. Also, in contrast to in the vulva, cog-1 has no comparable transcriptional impact on nhr-67 in ASER. cog-1 and nhr-67 therefore are striking examples of regulatory factors wired together in distinct configurations in different cell types (Sarin, 2009).

A nuclear receptor closely related to NHR-67, FAX-1, also controls neuronal identity and morphology in C. elegans. Similarly, its vertebrate homolog, PNR, promotes proper photoreceptor cell fate specification. In other species, the orthologs of NHR-67, Drosophila Tailless and vertebrate TLX, are also involved in various aspects of neuronal development. Tailless promotes the formation of large regions of the Drosophila brain by activating expression of the proneural gene lethal of scute, rendering the cells competent to form neural precursors. Tailless mutants result in the loss of several brain regions owing to a lack of neuroblast production. Similarly, vertebrate TLX is a key regulator of the cell cycle in neuronal stem cell populations. Tlx-/- mice are unable to maintain an undifferentiated population of stem cells and, consequently, lose several parts of outer brain regions. The finding of NHR-67 acting at several independent steps late in neuronal differentiation suggests that Tailless-like proteins in other organisms may also have late differentiation roles that might have been obscured by their earlier roles in neuronal precursor formation (Sarin, 2009).

A tissue-specific enhancer of the C. elegans nhr-67/tailless gene drives coordinated expression in uterine stem cells and the differentiated anchor cell

The nhr-67 nuclear receptor gene of C. elegans encodes the ortholog of the Drosophila tailless genes. In C. elegans, nhr-67 plays multiple roles in the development of the uterus during L2 and L3 larval stages. Four pre-VU cells are born in the L2 stage and form the precursor complement for the ventral surface of the mature uterus. One of the four pre-VU cells becomes the anchor cell (AC), which exits the cell cycle and differentiates, while the remaining three VU cells serve as stem cells that populate the ventral uterus. The nhr-67 gene functions in the development of both VU cell lineages and AC differentiation. Hypomorphic mutations in nhr-67 identify a 276bp region of the distal promoter that is sufficient to activate nhr-67 expression in pre-VU cells and the AC. The 276bp region includes 8 conserved potential cis-acting sites, including two E boxes and a nuclear receptor binding site. Mutational analysis demonstrates that the two E boxes are required for expression of nhr-67 in uterine precursor cells. The E/daughterless ortholog HLH-2 binds these sites as a homodimer, thus playing a central role in activating nhr-67 expression in the uterine precursors. The organization of the nhr-67 uterine precursor enhancer is compared to similar conserved enhancers in the egl-43, lag-2, and lin-3 genes, which contain the same HLH-2-binding E boxes and are similarly expressed in both pre-VU cells and the AC. This basic regulatory module allows the coordinated expression of at least four genes. Expression of genes in different cells that must coordinate to form a mature organ is driven by a shared set of promoter elements, which integrate multiple transcription factor inputs (Bodofsky, 2018).

Nuclear receptor TLX prevents retinal dystrophy and recruits the corepressor atrophin1

During mammalian embryogenesis, precise coordination of progenitor cell proliferation and differentiation is essential for proper organ size and function. The involvement of TLX (NR2E1), an orphan nuclear receptor, has been implicated in ocular development; Tlx^–/– mice exhibit visual impairment. Using genetic and biochemical approaches, this study shows that TLX modulates retinal progenitor cell proliferation and cell cycle re-entry by directly regulating the expression of Pten and its target cyclin D1. Additionally, TLX finely tunes the progenitor differentiation program by modulating the phospholipase C and mitogen-activated protein kinase (MAPK) pathways and the expression of an array of cell type-specific transcriptional regulators. Consequently, Tlx^–/– mice have a dramatic reduction in retina thickness and enhanced generation of S-cones, and develop severe early onset retinal dystrophy. Furthermore, TLX interacts with atrophin1 (Atn1), a corepressor that is involved in human neurodegenerative dentatorubral-pallidoluysian atrophy (DRPLA) and that is essential for development of multiple tissues. Together, these results reveal a molecular strategy by which an orphan nuclear receptor can precisely orchestrate tissue-specific proliferation and differentiation programs to prevent retinal malformation and degeneration (Zhang, 2006: full text of article).

The nuclear receptor tailless is required for neurogenesis in the adult subventricular zone

Tailless (Tlx) gene encodes an orphan nuclear receptor that is expressed by neural stem/progenitor cells in the adult brain of the subventricular zone (SVZ) and the dentate gyrus (DG). The function of Tlx in neural stem cells of the adult SVZ remains largely unknown. This study shows that in the SVZ of the adult brain Tlx is exclusively expressed in astrocyte-like B cells. An inducible mutation of the Tlx gene in the adult brain leads to complete loss of SVZ neurogenesis. Furthermore, analysis indicates that Tlx is required for the transition from radial glial cells to astrocyte-like neural stem cells. These findings demonstrate the crucial role of Tlx in the generation and maintenance of NSCs in the adult SVZ in vivo (Liu, 2008).

The subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG) are the largest germinal zones of sustained neurogenesis during adulthood in the mammalian central nervous system. Astrocyte-like type B cells in the adult SVZ are thought to be multipotent neural stem cells (NSCs). These cells give rise to transient amplifying type C cells, which in turn differentiate into type A cells (neuroblasts) that migrate to the olfactory bulb (OB) through the rostral migratory stream (RMS). The SVZ-OB system has recently been reported to also exist in the adult human brain (Liu, 2008).

Tailless is an orphan nuclear receptor, which is expressed in the periventricular neurogenic zone during mouse embryonic development. In the absence of this transcription factor, mutant animals survive, but suffer specific anatomical deficits in the cortex and the limbic system. Late-developing structures such as the upper cortical layers and the DG, are reduced in size. These phenotypic changes indicate that Tlx has an important role for brain development in the young postnatal stage. The loss of adult neurogenesis in Tlx-null mice does not exclude the possibility that this phenotype, lack of neurogenesis, is caused by the absence of Tlx during development. Recently, a study demonstrated that inactivation of Tlx leads to a decrease but not complete loss of neural stem cell proliferation in the DG. The role of Tlx in the adult neurogenic region of the SVZ, however, remains largely unknown. This study shows that the nuclear receptor Tlx is expressed with an unprecedented exclusivity in astrocyte-like B cells of the SVZ-OB system, and that it is a crucial determinant of the generation of adult NSCs in the SVZ and the SGZ. Tlx is essential for the maintenance of self-renewal of adult NSCs in the SVZ. This study also provides insights of the different identities of NSCs in the postnatal and adult brain, as well as in different neurogenic regions of the adult brain (Liu, 2008).

The nuclear receptor tailless induces long-term neural stem cell expansion and brain tumor initiation

Liu, H. K., et al. (2010). The nuclear receptor tailless induces long-term neural stem cell expansion and brain tumor initiation. Genes Dev. 24(7): 683-95. PubMed Citation: 20360385

Malignant gliomas are the most common primary brain tumors, and are associated with frequent resistance to therapy as well as poor prognosis. The nuclear receptor tailless (Tlx), which in the adult is expressed exclusively in astrocyte-like B cells of the subventricular zone, acts as a key regulator of neural stem cell (NSC) expansion and brain tumor initiation from NSCs. Overexpression of Tlx antagonizes age-dependent exhaustion of NSCs in mice and leads to migration of stem/progenitor cells from their natural niche. The increase of NSCs persists with age, and leads to efficient production of newborn neurons in aged brain tissues. These cells initiate the development of glioma-like

lesions and gliomas. Glioma development is accelerated upon loss of the tumor suppressor p53. Tlx-induced NSC expansion and gliomagenesis are associated with increased angiogenesis, which allows for the migration and maintenance of brain tumor stem cells in the perivascular niche. Tlx transcripts are overexpressed in human primary glioblastomas in which Tlx expression is restricted to a subpopulation of nestin-positive perivascular tumor cells. This study clearly demonstrates how NSCs contribute to brain tumorgenesis driven by a stem cell-specific transcription factor, thus providing novel insights into the histogenesis and molecular pathogenesis of primary brain tumors (Liu, 2010).